This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iron Regulatory Protein 1 (IRP1) and Iron Regulatory Protein 2 (IRP2), formerly referred to as iron-responsive element binding proteins (IRE-BPs). IRPs bind IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and prolongation of the half-life of the transferrin receptor mRNA. IRP1 is an iron-sulfur protein related to mitochondrial aconitase, a Krebs cycle enzyme, and it functions as a cytosolic aconitase in cells that are iron replete. In cells that are iron-depleted, IRP2 binds IREs with high affinity. Regulation of RNA binding activity involves a transition from a form of IRP1 in which a [4Fe-4S] cluster is bound, to a form that loses both iron and aconitase activity. The [4Fe-4S] containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster reveals that the physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status of the cluster appears to be the key to determining whether the protein will bind RNA. Little is known about the assembly and disassembly of iron-sulfur clusters in mammalian cells. In recent studies, we have identified mammalian genes homologous to genes that are implicated in bacterial iron-sulfur cluster assembly. Unlike IRP1, IRP2 is actively degraded in cells that are iron- replete. An additional exon present in IRP2 determines whether IRP2 is subject to degradation. This 73 amino acid exon is sufficient to confer upon IRP1 an iron -dependent degradation phenotype. Mutagenesis of cysteines within the 73 amino acid domain reveals that cysteines are required for degradation, and treatment of cells with proteasome inhibitors reveals that the proteasome is required for degradation. In order to approach questions about the physiology of iron metabolism, IRP1 has been ~knocked out~ in mice, using homologous recombination in embryonic cell lines. There is no obvious phenotype associated with loss of IRP1 function, and we speculate that both IRPs must be absent in order to see perturbations in iron metabolism. Accordingly, creation of IRP2 "knockouts" and double "knockouts" are underway.